Mutation Research, 120 (1983) 121-125 121 Elsevier Biomedical Press

Absence of a mutagenic effect after feeding 4 anti-cancer drugs to Drosophila melanogaster

Norman Todd", Julie Clements a, Pamela Zoeller a and Malcolm Phillips b

"Department of Biological Sciences, University of Exeter, Prince of Wales Road, Exeter, EX4 4PS (Great Britain) and bDepartment of Haematological Medicine, Musgrove Park Hospital, Taunton,

TA 1 5DA (Great Britain)

(Accepted 27 January 1983)

As a result of recent developments in anti-cancer chemotherapy, there have been such dramatic improvements in the treatment of leukaemias (Blacklock et al., 1981) that prolonged or complete remission is now being achieved with increasing regulari- ty. The follow-up of long-term survivors has, however, led to concern over possible late complications, particularly and perhaps most significantly, the apparent in- crease in the incidence of second malignancies (Coleman et al., 1977). In view of the suggested link between mutagenesis and carcinogenesis (see Hollstein et al., 1979), one explanation would seem to be that the drug treatment itself produces genetic changes which lead to further malignancies. Most studies on anti-cancer drugs have been concerned with their ability to cause cytogenetic damage, with only a few being screened for gene mutations. Thus, our laboratory is currently assessing the mutagenicity of 8 drugs (adriamycin, asparaginase, cyclophosphamide, cytosine arabinoside, 6-mercaptopurine, methotrexate, prednisolone and vincristine), both singly and in selected combinations, used regularly in the treatment of leukaemias in a mammalian cell assay and several yeast and Drosophila systems.

This report presents data on the ability of 4 of these drugs, cytosine arabinoside (CAS No. 147-94-4), methotrexate (CAS No. 59-05-2), vincristine (CAS No. 57-22-7) and prednisolone (CAS No. 50-24-8) to induce sex-linked recessive lethal mutations in Drosophila. Cytosine arabinoside is an anti-metabolite which inhibits DNA polymerase (Momparler, 1969) and is incorporated to a limited extent, into DNA (Creasey et al., 1968). Whilst there is no consistent data for it being mutagenic (see Huberman and Heidelberger, 1972), the drug can induce cytogenetic damage (Benedict and Jones, 1979). Methotrexate is a folic acid antagonist and was negative when tested for mutagenicity in bacteria (Seino et al., 1978) but capable of causing chromosome aberrations (Sieber and Adamson, 1975). Vincristine is a naturally- occurring plant alkaloid which binds to tubulin, the protein sub-unit of microtubules, preventing their polymerization. Not unexpectedly, by affecting spin-

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122

TABLE 1

THE SEX-LINKED RECESSIVE LETHAL TEST, MATERIALS AND METHODS

Source of the compounds

Way of administration

Preparation of test solution

Exposure time

Toxicity

Fertility

Genotype of treated males

Age of male at beginning of treatment

Age of male at mating

Genotype of parental female

Age of female at mating

Mating scheme

Brooding scheme

Scoring criteria in F2

Culturing temperature

Statistical methods

Vincristine sulphate Sigma Cytosine arabinoside Sigma Methotrexate Sigma Prednisolone sodium succinate Sigma

Feeding of adult males. Flies were treated in glass vials containing 15 filter paper squares saturated with l ml of test solution. The solutions were replenished daily.

The chemicals were dissolved in 5% sucrose solution. (In the case of methotrexate a pH adjustment was made using NaOH).

3 days.

Test chemicals were not toxic at the concentrations used.

Not noticeably affected.

Berlin K.

2-3 days.

Immediately after treatment, 5-6 days.

Basc (In (1)scSlLsc8R+ S,scSIscSwaB) (Lindsley and Grell, 1968).

2-5 days.

Standard Muller-5 scheme (Wiirgler et al., 1977).

The treated males were mated with fresh virgins for three con- secutive periods of 3, 2 and 2 days; 3 virgins for each mating and males mated individually.

F2 cultures with 2 or more wild-type males were considered as non- lethals. Retests were carried out to confirm lethality.

25°C.

2 × 2 contingency X 2 test.

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TABLE 2

FREQUENCIES OF SEX-LINKED RECESSIVE L E T H A L M U T A T I O N S AFTER T R E A T M E N T OF Drosophila melanogaster MALES W I T H VINCRISTINE OR CYTOSINE ARABINOSIDE

Treatment Broods

1 2 3 1-3

1 1 1 1 °7ol %1 °7ol %1

nchr nchr nchr nchr

5 2 5 12 Control s 0.19 0.10 0.20 0.17

2659 2052 2491 7202

1 0 2 3 Vincristine 0.16 - 0.28 0.16

612 541 707 1860 5 #g /ml 10 4 2 16 Vincristine b 0.26 0.12 0.06 0.15

3866 3360 3148 10374 10 ~g/ml 3 1 2 6

Vincristine 0.43 0.13 0.28 0.28 700 760 718 2178

15 /~g/ml 3 4 0 7 Cytosine 0.14 0.21 - 0.12 arabinoside c 2157 1942 1779 5878

1.0 m g / m l 1 1 3 5

Cytosine 0.04 0.04 0.14 0.07 arabinoside d 2753 2244 2196 7193

1.5 m g / m l 2 3 0 5

Cytosine 0.13 0.22 - 0.13 arabinoside e 1486 1376 941 3803

2.0 m g / m l

SPooled data f rom 4 Expts. , also presented in Table 3. bpooled data f rom 6 Expts.

CPooled data f rom 3 Expts. dpooled data from 4 Expts.

ePooled data f rom 2 Expts.

l, lethal; nchr, number o f chromosomes tested.

dle formation, the drug produces many chromosomal effects but it is in the main, not mutagenic (for review see Degraeve, 1978). Prednisolone is a synthetic analogue of the naturally-occurring corticosteroid hydrocortisone and virtually nothing is known about its genotoxicity.

Table 1 presents the materials and methods used in these experiments (after Kramers, 1977). The frequencies of sex-linked recessive lethal mutations obtained for control flies and flies fed on vincristine or cytosine arabinoside are given in Table 2 . It can be seen that neither drug increased the mutation frequency significantly above that observed in the control. The result with the spindle poison, vincristine, was not unexpected and it is interesting that one of us (J. Clements, un- published data) has found another such agent, colcemid, also to be negative in this system. The negative results for cytosine arabinoside are in agreement with the find-

124

TABLE 3

FREQUENCIES OF SEX-LINKED RECESSIVE LETHAL MUTATIONS AFTER TREATMENT OF Drosophila melanogaster MALES WITH METHOTREXATE OR PREDNISOLONE

Treatment Broods

1 2 3 1-3

1 1 1 1 0701 °701 0701 0701

nchr nchr nchr nchr

5 2 5 12 ControP 0.19 0.10 0.20 0.17

2659 2052 2491 7202

Methotrexate b 2 0.13 1 0.06 4 0.49 7 0.17 1514 1690 818 4022

10/~g /ml 1 2 0 3 Methotrexate b 0.05 0.14 - 0.07

2025 1389 934 4348 100 tLg/ml

1 0 0 1 Prednisolone O. 17 - - 0.06

587 615 508 1710 5 mg/ml 0 1 1 2

Prednisolone c - O. 11 O. 10 0.06 1282 948 992 3222

10 mg/ml 2 3 1 6 Prednisolone c O. 14 0.22 0.09 O. 16

1393 1378 1079 3850 20 mg/ml 1 1 2

Prednisolone 0.15 0.74 - - 0.25 650 136 786 50 mg/ml

apooled data from 4 Expts., also presented in Table 2. bpooled data from 3 Expts. CPooled data from 2 Expts. 1, lethal; nchr, number of chromosomes tested.

ings of F a h m y et al. (1966) who adminis tered the drug by inject ion. Likewise

negative results were ob ta ined for methotrexate and predniso lone (Table 3). In view

of the weakly positive results found for methotrexate in m a m m a l i a n cells (see

fol lowing letter), we are testing higher concent ra t ions of this drug in Drosophi la and

adminis te r ing it by in ject ion. To conclude, the da ta provide no evidence for any of

these 4 drugs being mutagenic . However , as several o f these c o m p o u n d s are k n o w n

to cause cytogenetic damage, fur ther work is being carried out using Drosophi la

systems designed to detect ch romosome breakage and loss.

A c k n o w l e d g e m e n t s

We thank Richard Ford , Mar t in Clarke and Ruper t Manley for their technical

assistance and express our grat i tude to the Cancer Research Campa ign and the

Musgrove Park Leukaemia Research Fund , T a u n t o n , for f inancia l support .

125

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